The study carried out in our laboratory demonstrated that protein restriction (low protein, LP) during fetal and neonatal life alters pancreatic development and impairs glucose tolerance later in life. In this study, we examined the role of the transcription factor Pdx1, a master regulator of β-cell differentiation and function along with its downstream target genes insulin, Glut2 and glucokinase (GK). The role(s) of these genes and protein products on the pancreata of male offspring from mothers exposed to LP diets were assessed during gestation, weaning, and adult life. Pregnant rats were allocated to two dietary treatments: control (C) 20% protein diet or LP, 8% protein diet. At birth, offspring were divided into four groups: C received control diet all life, LP1 received LP diet all life, LP2 changed the LP diet to C at weaning, and LP3 switched to C after being exposed to LP during gestation only. Body weights (bw) were significantly (P<0.001) decreased in all LP groups at birth. At weaning, only the LP3 offspring had their body weight restored to control levels. Pdx1 or any of the Pdx1-target genes were similar in all diets at day 21. However, at d130 Pdx1 mRNA expression and protein abundance were significantly decreased (P<0.05) in all LP groups. In addition, insulin mRNA and protein were decreased in LP1 and LP3 groups compared with C, Glut2 mRNA and GLUT2 protein levels were decreased in LP3 and GK did not change between groups. Intraperitoneal glucose tolerance test revealed impaired glucose tolerance in LP3 males, concomitant with decreased β-cell mass, islet area, and PDX1 nuclear protein localization. Collectively, this study suggests that restoring proteins in the diet after birth in LP offspring dramatically impairs glucose homeostasis in early adulthood, by altering Pdx1 expression and downstream-target genes increasing the risk to develop type 2 diabetes.